Project Summary/Abstract The developing human brain is susceptible to infection by several teratogenic neurotropic viruses, including Zika virus (ZIKV). In utero ZIKV infection can cause devastating abnormal brain development, highlighting large gaps in our understanding of viral infection during this critical phase of neurodevelopment. Recent studies have shown that ZIKV preferentially infects radial glia, astrocytes, and microglia and only rarely infects neurons. Yet little is known about secondary effects of infection on nearby, uninfected cells. In vivo, productively infected cells may represent a small minority of the tissue. In the developing brain in particular, paracrine effects on uninfected cells may disrupt the tightly regulated processes of neurogenesis, including proliferation, differentiation, and migration. This proposal investigates indirect effects of viral infection of the developing human brain. The response to viral infection in the brain includes important contributions from innate immunity in brain-resident cells, such as type I interferon (IFN) signaling. Despite IFN?s antiviral activity, human genetic conditions such as Aicardi-Goutires syndrome demonstrate that unmitigated type I IFN signaling can cause abnormal neurodevelopment. These paradoxical properties for IFN as both antiviral and neurotoxic raise an intriguing question: how is neurodevelopment affected by the innate immune response to a neurotropic virus such as ZIKV, which has devastating effects on the developing brain but seldom infects neurons? Preliminary data from single cell RNA sequencing are consistent with type I IFN stimulation in uninfected cells of ZIKV-infected brain tissue. Intriguingly, astrocytes and radial glia upregulate many type I IFN response genes, whereas neurons upregulate a selective subset, including ISG15. In human cells, it appears that ISG15 may limit the response to type I IFNs. The proposed research will test the hypothesis that during acute infection of the developing human brain, paracrine responses in uninfected cells contribute to the pathology of ZIKV infection and are naturally limited by ISG15 in especially vulnerable cells such as newborn neurons. Aim 1 will identify factors that mediate paracrine signaling from infected to uninfected cells, using organotypic slice culture of primary human fetal brain tissue with in vitro ZIKV infection. Aim 2 will dissect mechanisms for differential type I IFN response in primary human neurons versus glia. Experiments will address the possibilities of (i) baseline differences in signaling pathway components and (ii) differential chromatin accessibility. This aim will generate cell-type specific chromatin accessibility maps, opening new avenues for exploration of IFN responses in the developing brain. Aim 3 will characterize the role of ISG15 in uninfected neurons within an infected tissue. This approach may provide new insight into neuroprotective mechanisms. In summary, this project leverages single cell sequencing to launch an exploration into indirect effects of viral infection on the human developing brain. This work will shed light on an underexplored aspect of neurodevelopment at the intersection with infectious and autoimmune disease.